Optimization of the Weck-Cel Collection Method for Quantitation of Cytokines in Mucosal Secretions

Abstract

Measurement of immune components in mucosal secretions is important for the evaluation of local immunity at the mucosal surfaces. The Weck-Cel ophthalmic sponge provides a method for the collection of these secretions. The sponge absorbs a relatively large volume of material, therefore allowing for quantitation of multiple immune components. Additionally, it provides a method in which the same device may be used to collect specimens from different mucosal sites, such as the genital tract and oral cavity. This sampling technique has successfully been applied for collection and measurement of antibody in oral and genital tract secretions. The purpose of this work was to optimize the extraction of protein from the sponge matrix. Of particular interest was the recovery of cytokines from the sponge. Satisfactory recovery of the cytokines interleukin 1␤ (IL-1␤), IL-2, IL-5, IL-12, IL-6, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor was obtained. However, IL-4 and gamma interferon recovery rates remained low. Using an alteration of the published extraction method, cytokine concentrations were measured in cervical secretions from women using oral contraceptives. The data revealed detectable concentrations of IL-6, IL-10, IL-8, and IL-12 on cycle days 9 and 20. The proposed technique provides an easy, practical, and consistent method for collection of nonconventional body fluids, such as cervicovaginal fluids and saliva, for the assay of immunoglobulins and several cytokines.Monitoring or analysis of humoral immune components in conventional body fluids, such as blood and urine, involves common methods. However, measurement of these factors in nonconventional biological fluids, such as cervical secretions, vaginal fluid, and saliva, is complex. The difficulties lie not only in the analysis of these mucosal fluids but also in obtaining reproducible and unaltered samples. The accuracy and consistency of the sampling procedures can ultimately affect experimental outcomes and quantitation of the individual components. Yet the assessment of such fluids is important because it gives insight into the local immune response, physiological modifications induced by infection, and potential drug profiles at the site of action (11,12).There are a number of different methods available for the collection of genital tract secretions, such as cervicovaginal washes, aspiration, and Wick absorption. Each technique has proven its utility but also has a downside. The washes yield a significant amount of material but combine vaginal and cervical secretions. These two secretions have different roles in the protection of the genital tract, and combining them prevents studies of each secretion. Aspiration works well for collection of cervical secretions at midcycle in women who are ovulating but yields little volume at other times in the cycle or when women are using oral contraceptives. Finally, the Wicks collect cervical and vaginal secretions individually but absorb a very small amount of material. The collection volume li...

Comparison of Ophthalmic Sponges for Measurements of Immune Markers from Cervical Secretions

“…In in vitro experiments, we confirmed that IL-4 and IFN-␥ may be poorly recovered from Weck-Cel ophthalmic sponges (16) and demonstrated that immune markers may be more reliably recovered from in vitro-loaded Ultracell and Merocel ophthalmic sponges. In particular, recovery from Merocel sponges, which are composed of hydroxylated polyvinyl acetate, was nearly 100% for all tested cytokines in the in vitroloading experiment, except for IL-6, whose levels of recovery were equally poor from all sponges.…”

“…Sponges were extracted according to a previously described protocol (16). Briefly, each sponge was weighed to estimate the volume of secretions absorbed onto the sponge.…”

“…We have successfully implemented these collections in our large cohort study since 1996 (2). However, a recent study to optimize recovery of immune markers from Weck-Cel sponges demonstrated that some immune markers, such as interleukin-4 (IL-4) and gamma interferon (IFN-␥), were poorly recovered from in vitro loading of these sponges (16). The reason for poor recovery of these cytokines is uncertain, but one possible explanation is that specific immune markers irreversibly bind to the absorbent material of this sponge.…”

Effect of storage temperatures on the stability of cytokines in cervical mucous

“…We used the protein extraction protocol described [12]. Briefly, the sponges were equilibrated in 300 l of extraction buffer consisting of PBS (pH 7.0), 0.25M NaCl and 10% fetal calf serum, for 30 min at 4°C.…”

Influence of Exogenous Reproductive Hormones on Specific Antibody Production in Genital Secretions after Vaginal Vaccination with Recombinant Cholera Toxin B Subunit in Humans

“…Cervical secretions were obtained with a syringe, whereas vaginal fluids were collected with Weck-cel sponges. Cervical secretions are not regarded as ideal specimens for assessment of genital immune responses in large-scale immunization studies, since the collection is timeconsuming, requires gynecological assistance, and at times yields small amounts of secretions (9,28). Vaginal fluid, on the other hand, is an easily accessible secretion.…”